Proteins are important components of cells and their activities determine various cellular functions. All diseases associate with some changes in protein expressions; therefore, comparison of the protein expressions between normal and abnormal biological samples are useful for understanding disease mechanisms and clinical diagnostics.
Proteins can be detected by immunological methods, such as Western blotting and Enzyme-Linked Immunosorbent Assay (ELISA). Western blotting (Immunoblotting) is a widely used technique in protein research. It combines the resolution of gel electrophoresis with the specificity of immunochemical detection and is powerful in determining a number of important characteristics of protein antigens, e.g., the relative molecular weight and the quantity of an antigen in a protein sample. When combined with immunoprecipitation, Western blotting allows the detection of specific interactions between proteins. It is also useful in detecting protein posttranslational modifications, e.g., protein tyrosine phosphorylation (Kamps, 1991. Methods Enzymol 201:101-10). Protein arrays have been used for examining protein expression, protein phosphorylation, protein-protein interaction, protein-DNA interaction, and protein-analyte interaction (Lueking et al. 1999 Anal. Biochem. 270, 103-111. Wang et al. 2000 Mol. Cell Biol. 20, 4505). Immunochemical staining is another versatile technique in determining both the presence and localization of proteins (Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Press, 1988). An antibody array-based staining method was also developed for examining protein expression, protein cellular and subcellular localization, and other properties (Wang, 2004. Proteomics 4, 20-26).
Many protein immunodetection methods involve the interaction of a reagent (e.g. antibody) and a ligand (e.g. a protein) immobilized on a solid support, such as a membrane or a multi-well plate. In most cases, the binding occurs during the incubation of the ligand-bound support in the reagent solution. For example, a standard procedure for Western blotting includes the steps of separating proteins by gel electrophoresis, transferring proteins from a gel to a membrane support, and sequential incubation of the blot membrane in blocking, washing, and antibody solutions.
In general, the immunodetection is a process involving multiple changes of solutions, and is usually extended over several hours. A key but time-consuming step is the binding of an antibody to its antigen. Several factors influence the time taken to reach binding equilibrium, including the rate of diffusion and the affinity of the antibody for the antigen. It is desirable to accelerate the process to save time.
After their formation, the antibody-antigen complexes should be maintained till the end of the assay. However, because antibody-antigen interactions are reversible, the subsequent washing and incubation could lead to significant dissociation of the antibody-antigen complexes. Therefore, it is also critical to shorten the assay process in order to maximize the antibody-antigen binding.
Accelerated assay procedure is desirable in many applications. For example, a fast assay format makes it possible to use the assay in applications requiring results in rush. Several strategies have been used to facilitate antibody-antigen binding and shorten immuno-detection process. For example, agitation, usually by shaking, is routinely used to accelerate antibody diffusion, thereby its binding to antigen.
A vacuum-assisted filtration-like process was used in The SNAP i.d.™ system from Millipore, which claims that it can shorten the Western blotting procedure from several hours to less than 45 minutes. Filtration is a commonly used method for the separation of solids from fluids by passing the fluids through filter(s), such as a porous membrane. Oversize solids in the fluid are retained. Filtration devices comprising various components are well known. Many device designs have been made for filtration process; widely used to clarify and sterilize biological solutions, such as fetal calf serum, tissue culture media and the like. Vacuum-assisted filtration-like process has been used in immobilizing proteins and nucleic acids on a membrane support; and in purifying DNA. For examples, the 96-well Bio-Dot® and 48-well Bio-Dot SF microfiltration units from Bio-Rad are used for binding proteins or nucleic acids in solution onto membranes.
Besides SNAP i.d, several other prior arts also described methods of passing fluid through a membrane in immunoassays. For example, U.S. Pat. Nos. 4,366,241 and 4,632,901 describes using the capillary action of an absorbent material to draw fluids through a reaction-supporting membrane. U.S. Pat. No. 5,155,049 described another technique for passing liquid through a membrane.
There are limitations when a filtration-like process is used in reagent-ligand binding. Particularly, because one passage is usually insufficient for maximum binding, the existing methods do not provide optimal conditions for maximum binding. Therefore, there is a need for improved methods to increase binding.
In many applications it is usually necessary to probe multiple sample membranes. For example, it is often necessary to probe multiple western blot membranes when a large number of protein samples are studied. In clinic applications, it is often needed to screen many patient samples for the same biomarker. Therefore, there is a need for fast, easy simultaneous detection of ligands on multiple porous membranes.